KAPA Stranded mRNA-Seq Kit

Protocol tips

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	This protocol has been validated for library construction from 100 ng – 4 μg intact, total RNA, in ≤50 µL of RNase-free water.

Protocol tips
This protocol has been validated for library construction from 100 ng – 4 μg intact, total RNA, in ≤50 µL of RNase-free water.

Publication protocol

Primary chondrocytes of Col11a2-CreER; Sik3flox/flox mice were cultured in DMEM, 10% FBS medium for 5 days on a culture dish. Then, the chondrocytes were centrifuged and incubated in tubes for 3 days. The resulting cell pellets were incubated in the same medium in the presence or absence of 300 μM pterosin B for another 4 weeks. RNAs were extracted from the cultured chondrocyte pellets using QIAzol Lysis Reagent (Qiagen) and miRNeasy Mini Kit (Qiagen). RNAs were also extracted from various mouse tissues.
A 100 ng of total RNA was subjected to library preparation using KAPA stranded mRNA-seq Kits (KAPA Biosystems, Woburn, MA), according to the manufacturer's instruction. The libraries were sequenced in 100-cycle single-read mode of HiSeq2500. All sequence reads were extracted in FASTQ format using BCL2FASTQ Conversion Software 1.8.4 in the CASAVA 1.8.2 pipeline. The sequence reads were mapped to mm9 reference genes, using Tophat v2.0.14. Gene expression values were calculated and normalized using rpkmforgenes (10 December 2012). Averages of gene expression values from triplicate cultured chondrocyte pellet samples were calculated. Two-way hierarchical clustering of gene expressions in cultured chondrocyte pellets (n=3) and tissues (n=1) was conducted using the Euclidian distance and the ward method of hclust function in R3.2.1.

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